Botulinum neurotoxins (BoNTs) are widely used in medicine to treat a variety of neuromuscular disorders, taking advantage of their inhibitory effect on motor-endplate transmission after local injection and leading to localized muscle paralysis. It has become apparent that BoNTs can also enter other neuronal cells including sensory and CNS neurons. A robust block of neuraxial primary afferent and second order neurons processing can be achieved by direct spinal intrathecal delivery of the toxin. We (and others) have shown that such intrathecal delivery of BoNTs can produce a potent and persistent, but reversible, block of central nociceptive processing in a variety of pain models. However, the therapeutic utility of this approach is limited by the fact that the targeting and uptake of holotoxins is not specific to neuronal subtype. Thus the possibility of accompanying muscle paralysis and facilitated sensory processing due to block of inhibitory interneurons is of paramount importance. General effects would result in a heightened pain state rather than relief from pain, and has in fact once been described after an accidental injection of BoNT/A into the intrathecal space of a patient. We are proposing to overcome this detriment by targeting the enzymatic portion of the BoNT light chain (LC) specifically to terminals and neurons involved in pain processing. We hypothesize that stable and targeted BoNT derivatives can be created by coupling of recombinant truncated version of the BoNT LC to substance P or the peptide DAMGO, and that these derivatives will provide a long lasting and yet reversible block of nociceptive processing. Past efforts to create such constructs have been hindered by the postulate that BoNT LC cannot enter cells without possessing regions of BoNT heavy chain. Our innovative approach uses G- protein coupled receptor driven internalization after specific binding of the sensory neuron ligands to their respective receptors, such that the heavy chain is not needed for internalization. Our preliminary data and recently that of others with sP demonstrates the feasibility of this approach. We propose here to create a series of constructs using light chains of different BoNT serotypes (which differ in their longevity enabling regulation of duration of drug action) and at least two different ligands (substance P and DAMGO), which bind to the NK1and mu opioid receptors, respectively. These receptors are distributed on spinal terminals /cell bodies known to play a central role in pain processing. Cell models will examine selective entry into neurons expressing these receptors, and animal models will assess effects of the constructs on nociception and establish therapeutic ratios (e.g. analgesic /side effect doses). The use of intrathecal toxins has been validated with agents such as sP-saporin and resiniferatoxin, which are in clinical trials for chronic pain patients. But, unlike these agents, he targeted LCs can produce blocks of high selectivity and of varying durations of action. The results from this project have the potential to improve treatment options for chronic pain. The constructs created in this project will also provide tools for future studies of pain processing pathways. 1